A transcriptional network governing ceramide homeostasis establishes a cytokine-dependent developmental process

Transcriptional mechanisms controlling developmental processes establish and maintain proteomic networks, which can govern the levels of intracellular small molecules. Although dynamic changes in bioactive small molecules can link transcription factor and genome activity with cell state transitions, many mechanistic questions are unresolved. Using quantitative lipidomics and multiomics, we discover that the hematopoietic transcription factor GATA1 establishes ceramide homeostasis during erythroid differentiation by regulating genes encoding sphingolipid metabolic enzymes. Inhibiting a GATA1-induced sphingolipid biosynthetic enzyme, delta(4)-desaturase, or disrupting ceramide homeostasis with cell-permeable dihydroceramide or ceramide is detrimental to erythroid, but not myeloid, progenitor activity. Coupled with genetic editing-based rewiring of the regulatory circuitry, we demonstrate that ceramide homeostasis commissions vital stem cell factor and erythropoietin signaling by opposing an inhibitory protein phosphatase 2A-dependent, dual-component mechanism. Integrating bioactive lipids as essential components of GATA factor mechanisms to control cell state transitions has implications for diverse cell and tissue types.

1. Treatment of cells with 4HPR leads to changes in levels of Cer and dhCer species (Fig 1 G and H).Do the corresponding ceramide synthase genes change expression as well? 2. Treating G1E-ER4 cells with 4HPR blocks the increase in EpoR expression (Figure 2E).Can you rule out that the effects of 4HPR on erythroid differentiation are not due to defects in EpoR expression? 3.In extended figure 2D, it looks like 4HPR blocks the progression from CD71-Ter119-to CD71+Ter119-cells, is that also true if you treat mouse of human primary erythroid progenitors with 4HPR? 4. Does C6-cer or another ceramide rescue G1E-ER4 cells treated with 4HPR? 5. Treating cells with C6-cer also inhibits erythroid development, does this C6-cer get metabolized into downstream products such as Sphingosine?How do you rule out that the effects of C6-cer are not caused in part by downstream metabolites?6.The signaling experiments are crucial to the conclusions of the paper, but with the exception of Figure 6K, none of the blots show levels of EpoR, p-EpoR, Kit and p-Kit.Given that 4HPR decrease EpoR expression, these controls should be included.7. Do you get a shift in the dose response to Epo in colony formation when cultures are treated with C6-Cer? 8.An explanation of how the bacterial sphingomyelinase works would be helpful.There is no description of this experiment in the methods.
Reviewer #2 (Remarks to the Author): The ceramide pathway was previously shown to inhibit erythropoiesis in various models, including human hematopoietic stem cells, whereas sphingosine-1 phosphate restored erythroid differentiation.Ceramides (Cer) were shown to trigger granulomonocytic differentiation.These events were already correlated to the modulation of GATA-1/2 transcription factors.Moreover, ceramides were shown to modulate the mTOR/AKT pathway leading to inhibition of autophagy required for erythroid differentiation.
Here Liao et al. further extend this knowledge by establishing a link between GATA-1 and the gene battery required for Cer metabolism by using a transcriptome/proteome approach.They conclude that GATA1 regulation of sphingolipid metabolic genes contributes to physiological ceramide homeostasis, essential for erythroid differentiation.They clearly demonstrated that Cer homeostasis is essential for erythroblast function and differentiation.Subsequently, they disrupted Cer homeostasis to yield excessive dhCer or Cer inhibiting erythroid progenitors in line with previously published data.This disruption modulates cytokine signaling systems essential for erythroid progenitor.In addition, they discovered Ormdl3 an upstream regulator of ceramide synthesis.Eventually, they show that Cer interferes with Epo and SCF receptors.
Whereas the paper is interesting in the field of erythroid differentiation, a number of improvements need to be provided.
1.The authors use a well-known mouse G1E cell model for most of their investigations.The authors are asked to validate key experimental results with erythropoietin-differentiated human hematopoietic stem cells to confirm the relevance of the mouse results.
2. Investigation of viability in Figure 2C.The authors are asked to indicate viability in % and not as a relative unit compared to control.Extended data figure 2A shows Facs results of AnnexinV/PI staining.This analysis is likely artefactual as the voltage in FSC versus SSC was probably erroneously set, leading to a distortion of the result.This representation/experiment needs to be considerably improved/redone.The same figure shows that the cells were largely dying, even without treatment.64% of viability as a starting point hints at sensitization of the cells.How do the authors explain this low viability and the impact on the outcome of the treatments? 3.In Extended data Figure 5, the authors observe a significantly reduced SCF-induced p-AKT and Epoinduced p-AKT and p-STAT5.As the WB in the manuscript only shows a modest variation, the authors are asked to present the original unaltered triplicates.The lipidomics methodology, targeted quantification of sphingolipids and untargeted lipidomics, applied in this study are well described and suitable to main purpose of the manuscript.However, in order to help readers and future similar experiments I do recommend to: -add a brief method description containing the most relevant information for sphingolipids quantification.This information should contain a table with sphingolipid species targeted, retention time, transitions monitored and internal standard use for quantification.
-deposit the data obtained from the untargeted lipidomics approach, including MS and MS/MS spectra, in a public data repository platform like Metabolomics Workbench.

Other comments:
In the excel file provided as attached file there is information about sphingolipids quantification.This information has not been formatted and it is reported as exported from the used quantification software.Only if the readers are familiar with this type of data can be understand.So I do recommend to just keep the summarized table containing the concentration levels.On the other hand, in this table there is only one dihydroceramide (DhCer C16:0) but in Figure 1H the heatmap contains other DhCer species.Where these other DhCer targeted and quantified together with other sphingolipids?treatment or C6-Cer and 4HPR double treatment abrogated Epo-dependent phosphorylation of AKT and ERK.C6-Cer and 4HPR double treatment further reduced Epo-induced p-STAT5 in comparison to a single treatment.These results suggest that ceramide does not rescue the impact of 4HPR on cytokine signaling, further strengthening our conclusion that accumulation of dhCer or Cer is detrimental to erythroid cells.5) Query: "Treating cells with C6-cer also inhibits erythroid development, does this C6-cer get metabolized into downstream products such as Sphingosine?How do you rule out that the effects of C6-cer are not caused in part by downstream metabolites?" Response: C6-Cer can be incorporated into sphingolipid metabolic pathways and metabolized into downstream products, like sphingosine, endogenous long-chain ceramides, and sphingomyelin.We demonstrated that inhibition of cytokine signaling by C6-Cer depends on PP2A activity (Fig. 5), and prior biochemical analyses by the Hannun laboratory demonstrated that either short-chain or longchain ceramide bind PP2A and enhance its activity in vitro (Dobrowsky, et al., J. Biol.Chem., 1993; Chalfant et al., J. Biol.Chem., 1999).Since no other sphingolipid species have been reported to enhance PP2A activity in vitro, we believe that the PP2A-dependent C6-Cer activity is primarily due to increased intracellular ceramide levels.
6) Query: "The signaling experiments are crucial to the conclusions of the paper, but except for Figure 6K, none of the blots show levels of EpoR, p-EpoR, Kit and p-Kit.Given that 4HPR decrease EpoR expression, these controls should be included." Response: We thank the reviewer for highlighting the need to analyze KIT and EPOR activation in the signaling experiments.We conducted new experimentation to measure KIT and EPOR phosphorylation upon acute (30 min) or prolonged (4 h) disruption of ceramide homeostasis by 4HPR, C6-dhCer, or C6-Cer.These analyses revealed that a 4 h treatment with 4HPR, C6-dhCer, or C6-Cer reduced SCF-dependent KIT phosphorylation and Epo-dependent EPOR phosphorylation (Fig. 4j-m), while acute C6-Cer treatment reduced Epo-dependent EPOR and JAK2 phosphorylation without impacting p-KIT (Fig. 6k-n).These results provide further evidence for our model that a dualcomponent mechanism mediates Cer/PP2A activity to regulate cytokine signaling (Fig. 6o).7) Query: "Do you get a shift in the dose response to Epo in colony formation when cultures are treated with C6-Cer?" Response: In Fig. 3c, we demonstrated that C6-Cer almost completely abrogated erythroid colonies (CFU-E and BFU-E) in M3434 media that contains maximal level of Epo.Therefore, one would expect to see little to no erythroid colonies for cultures treated with C6-Cer in response to sub-maximal levels of Epo.

4 .
Minor: Line 285 and Extended data figures explain the acronym PDBE.Reviewer #3 (Remarks to the Author): Comments to the manuscript entitle "Transcriptional Network Governing Ceramide Homeostasis Establishes a Cytokine-Dependent Developmental Process".